System and method for a biomimetic fluid processing

ABSTRACT

A system and method are provided for harvesting target biological substances. The system includes a substrate and a first and second channel formed in the substrate. The channels longitudinally extending substantially parallel to each other. A series of gaps extend from the first channel to the second channel to create a fluid communication path passing between a series of columns with the columns being longitudinally separated by a predetermined separation distance. The system also includes a first source configured to selectively introduce into the first channel a first biological composition at a first channel flow rate and a second source configured to selectively introduce into the second channel a second biological composition at a second channel flow rate. The sources are configured to create a differential between the first and second channel flow rates to generate physiological shear rates along the second channel that are bounded within a predetermined range.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/758,915 filed Jul. 1, 2015, which is the U.S. National StageApplication of International Patent Application PCT/US2013/070910 filedNov. 20, 2013, which claims the benefit of, and incorporates herein byreference in its entirety, U.S. Provisional Patent Application61/848,424 filed Jan. 3, 2013.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 1K99HL114719-01A1awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present disclosure generally relates to fluid systems, includingmicrofluidic devices, systems that include such devices, and methodsthat use such devices and systems. More particularly, the presentdisclosure relates to devices, systems, and methods for generatingfunctional biological material, substances, or agents based onbiomimetic platforms.

Blood platelets (PLTs) are essential for hemostasis, angiogenesis, andinnate immunity, and when numbers dip to low levels, a condition knownas thrombocytopenia, a patient is at serious risk of death fromhemorrhage. Some causes for low platelet count include surgery, cancer,cancer treatments, aplastic anemia, toxic chemicals, alcohol, viruses,infection, pregnancy, and idiopathic immune thrombocytopenia.

Replacement PLTs to treat such conditions are generally derived entirelyfrom human donors, despite serious clinical concerns owing to theirimmunogenicity and associated risk of sepsis. However, the shortagescreated by increased demand for PLT transfusions, coupled withnear-static pool of donors and short shelf-life on account of bacterialtesting and deterioration, are making it harder for health careprofessionals to provide adequate care for their patients. Moreover,alternatives such as artificial platelet substitutes, have thus farfailed to replace physiological platelet products.

In vivo, PLTs are produced by progenitor cells, known as megakaryocytes(MKs). Located outside blood vessels in the bone marrow (BM), MKs extendlong, branching cellular structures (proPLTs) into sinusoidal bloodvessels, where they experience shear rates and release PLTs into thecirculation. While functional human PLTs have been grown in vitro, cellculture approaches to-date have yielded only about 10 percent proPLTproduction and 10-100 PLTs per human MK. By contrast, nearly all adultMKs in humans must produce roughly 1,000-10,000 PLTs each to account forthe number of circulating PLTs. This constitutes a significantbottleneck in the ex vivo production of platelet transfusion unit.Although second generation cell culture approaches have provided furtherinsight into the physiological drivers of PLT release, they have beenunable to recreate the entire BM microenvironment, exhibiting limitedindividual control of extracellular matrix (ECM) composition, BMstiffness, endothelial cell contacts, or vascular shear rates; and havebeen unsuccessful in synchronizing proPLT production, resulting innon-uniform PLT release over a period of 6-8 days. Moreover, theinability to resolve proPLT extension and release under physiologicallyrelevant conditions by high-resolution live-cell microscopy hassignificantly hampered efforts to identify the cytoskeletal mechanics ofPLT production to enable drug development and establish new treatmentsfor thrombocytopenia. Therefore, an efficient, donor-independent PLTsystem capable of generating clinically significant numbers offunctional human PLTs is necessary to obviate risks associated with PLTprocurement and storage, and help meet growing transfusion needs.

Considering the above, there continues to be a clear need for devices,systems, and methods employing platforms that can recapitulate vascularphysiology to accurately reflect the processes, mechanisms, andconditions influencing the efficient production of functional humanblood platelets. Such platforms would prove highly useful for thepurposes of efficiently generating human platelets for infusion, as wellas for establishing drug effects and interactions in the preclinicalstages of development.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding a biomimetic fluidic system and method, for example, forgenerating functional human blood platelets using a platformrepresentative of physiologically accurate conditions, environments,structures, and dynamic flows. The approach is amenable for infusivetreatment of platelet-deficient conditions, such as thrombocytopenia, aswell as for drug development applications.

In accordance with one aspect of the present invention, a biomimeticmicrofluidic system is provided that includes a substrate. The systemalso includes a first channel formed in the substrate that extends froma first input to a first output and along a longitudinal dimension andextends along a first transverse dimension. The system also includes asecond channel formed in the substrate that extends from a second inputto a second output along the longitudinal dimension and extends along asecond transverse dimension. The first and second channels extendsubstantially parallel. The system further includes a series of gapsextending from the first channel to the second channel to create a fluidcommunication path passing between a series of columns. The columns arelongitudinally separated by a predetermined separation distance.Notably, the predetermined distance may be uniform or may vary within arange of predetermined distances such that the gaps have varying widths.The system also includes a first source connected to the first input andconfigured to selectively introduce into the first channel at least onefirst biological composition at a first channel flow rate. The systemnext includes a second source connected to the second input andconfigured to selectively introduce into the second channel at least onesecond biological composition at a second channel flow rate. The firstchannel flow rate and the second channel flow rate create a differentialconfigured to generate physiological shear rates along the secondchannel bounded within a predetermined range and to influence the flowwithin the first channel through the series of gaps.

In another aspect of the present invention, a method is disclosed forproducing a physical model of at least one of a bone marrow and bloodvessel structure. The method includes providing a biomimeticmicrofluidic system that includes a substrate and a first channel formedin the substrate that extends from a first input to a first output alonga longitudinal dimension and extends along a first transverse dimension.The system also includes a second channel formed in the substrate thatextends from a second input to a second output along the longitudinaldimension and extends along a second transverse dimension. The first andsecond channels extend substantially parallel. The system furtherincludes a series of gaps extending from the first channel to the secondchannel to create a fluid communication path passing between a series ofcolumns. The columns are longitudinally separated by a predeterminedseparation distance. The system also includes a first source connectedto the first input and a second source connected to the second input.The method includes introducing the first biological substance into theupper channel at a first channel flow rate using the first source andintroducing the second biological substance into the lower channel at asecond channel flow rate using the second source to create adifferential between the first and second channel flow rates to generatephysiological shear rates along the second channel that are boundedwithin a predetermined range. The method also includes harvesting atarget biological substance produced proximate to the gaps by thephysiological shear rates.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will hereafter be described with reference to theaccompanying drawings, wherein like reference numerals denote likeelements.

FIG. 1 is a schematic illustration of a biomimetic microfluidic systemin accordance with the present invention.

FIG. 2A shows microscopy images depicting a coating of each microfluidicchannel with bone marrow and blood vessel proteins to reproduceextra-cellular matrix (ECM) composition, in accordance with the presentinvention.

FIG. 2B shows microscopy images depicting megakaryocytes (MKs) trappedin gaps or microchannels selectively embedded in alginate gel (whitearrow), modeling 3-dimensional ECM organization and physiological bonemarrow (BM) stiffness, in accordance with the present invention.

FIG. 2C shows microscopy images of human umbilical vein endothelialcells (HUVECs) selectively cultured in the fibrinogen-coated secondchannel to produce a functional blood vessel, in accordance to thepresent invention.

FIG. 2D shows a combined image of the complete system.

FIG. 2E is a graphical depiction of a simulated distribution of shearrates within a biomimetic microfluidic system in accordance with thepresent invention.

FIG. 2F is a graphical depiction of shear rates as a function oftransverse (axial) distance from first channel for several infusionrates, in accordance with the present invention.

FIG. 2G is a graphical depiction of shear rates as a function of thenumber of block microchannels (slits or pores), in accordance with thepresent invention.

FIG. 3A is a graphical of depiction the diameter distribution forcultured MKs at 0 and 18 hours, in accordance with the presentinvention.

FIG. 3B shows microscopy images of MKs in static culture illustratingthe production of proPLTs at 6 hours post-purification, in accordancewith the present invention.

FIG. 3C shows microscopy images of MKs under physiological shearillustrating the production proPLTs immediately upon trapping, inaccordance with the present invention.

FIG. 3D is a graphical depiction of an increased percentage ofproPLT-producing MKs under physiological shear over those of staticcultures, in accordance with the present invention.

FIG. 3E is a graphical depiction illustration that proPLT extensionrates under physiological shear are increased significantly as comparedto static cultures, in accordance with the present invention.

FIG. 4A shows microscopy images illustrating MKs squeezing through 3μm-wide microchannels, supporting a model of vascular PLT production, inaccordance with the present invention.

FIG. 4B shows microscopy images illustrating MKs extending largefragments through 3 μm-wide microchannels, supporting a model ofvascular PLT production, in accordance with the present invention.

FIG. 4C shows microscopy images illustrating proPLT extension, inaccordance with the present invention.

FIG. 4D shows microscopy images illustrating proPLT extension andabscission events at different positions along the proPLT shaft, inaccordance with the present invention.

FIG. 4E shows microscopy images illustrating that following abscission,the resulting proPLT end formed a new PLT-size swelling at the tip,which was subsequently extended and released, with the cycle repeated,in accordance with the present invention.

FIG. 4F is a graphical depiction illustrating that increased shear rateswithin physiological ranges do not increase proPLT extension rate, inaccordance with the present invention.

FIG. 4G shows miscrocopy images illustrating that MKs retrovirallytransfected to express GFP-β1 tubulin showed proPLT extensions and werecomprised of peripheral MTs that form coils at the PLT-sized ends, inaccordance with the present invention.

FIG. 4H is a graphical depiction illustrating that 5 μM Jasplankinolide(Jas, actin stabilizer) and 1 mM erythro-9-(3-[2-hydroxynonyl] (EHNA,cytoplasmic dynein inhibitor) inhibit shear-induced proPLT production,in accordance with the present invention.

FIG. 4I shows microscopy images illustrating drug-induced inhibition ofproPLT production under physiological shear, in accordance with thepresent invention.

FIG. 5A is a graphical depiction illustrating that microfluidicdevice-derived mFLC-PLTs manifest structural and functional propertiesof blood PLTs, in accordance with the present invention.

FIG. 5B is a graphical depiction illustrating biomarker expression, andforward/side scatter and relative concentration of GPIX+ mFLC-MKsinfused into the microfluidic device following isolation on culture day4, and effluent collected from the microfluidic device 2 hours postinfusion, in accordance with the present invention.

FIG. 5C is a graphical depiction illustrating that the application ofshear shifts GPIX+ produce toward more PLT-sized cells relative tostatic culture supernatant, in accordance with the present invention.

FIG. 5D shows microscopy images illustrating that in the microfluidicdevice, mFLC-MKs are converted into PLTs over a period of 2 hours, inaccordance with the present invention.

FIG. 5E is a graphical depiction illustrating that the application ofshear shifts product toward more PLT-sized β1 tubulin+ Hoescht-cellsrelative to static culture supernatant, in accordance with the presentinvention. The insert shows quantitation of free nuclei in the effluent.

FIG. 5F shows microscopy images illustrating that microfluidicdevice-mPLTs are ultrastructurally similar to mouse blood PLTs andcontain a cortical MT coil, open canalicular system, dense tubularsystem, mitochondria, and characteristic secretory granules, inaccordance with the present invention.

FIG. 5G shows microscopy images illustrating that microfluidicdevice-mPLTs and PLT intermediates are morphologically similar to mouseblood PLTs and display comparable MT and actin expression, in accordancewith the present invention.

FIG. 6A is a graphical depiction illustrating that microfluidicdevice-derived hiPSC-PLTs manifest structural and functional propertiesof blood PLTs, where hiPSC-MKs reach maximal diameter (20-60 μm) onculture day 15, in accordance with the present invention

FIG. 6B shows a microscopy image illustrating that hiPSC-MKs areultrastructurally similar to primary human MKs and contain a lobulatednuclei, invaginated membrane system, glycogen stores, organelles, andcharacteristic secretory granules, in accordance with the presentinvention.

FIG. 6C shows microscopy images illustrating that hiPSC-MKs in staticculture begin producing proPLTs at 6 hours post-purification, and reachmaximal proPLT production at 18 hours, in accordance with the presentinvention.

FIG. 6D shows a microscopy image illustrating that hiPSC-MKs underphysiological shear (˜500 s⁻¹) begin producing proPLTs immediately upontrapping and extend/release proPLTs within the first 2 hours of culture,in accordance with the present invention.

FIG. 6E is a graphical depiction illustrating that percentproPLT-producing hiPSC-MKs under physiological shear are increasedsignificantly over static cultures, in accordance with the presentinvention.

FIG. 6F is a graphical depiction illustrating that proPLT extensionrates under physiological shear are ˜19 μm/min, in accordance with thepresent invention.

FIG. 6G shows microscopy images illustrating that microfluidic devicederived-hPLTs are ultrastructurally similar to human blood PLTs andcontain a cortical MT coil, open canalicular system, dense tubularsystem, mitochondria, and characteristic secretory granules inaccordance with the present invention. Top-right insert shows peripheralMT coil.

FIG. 6H shows microscopy images illustrating that microfluidic devicederived-hPLTs are morphologically similar to human blood PLTs anddisplay comparable MT and actin expression, in accordance with thepresent invention.

FIG. 6I shows microscopy images illustrating that microfluidic devicederived-mPLTs form filpodia/lamellipodia on activation and spread onglass surface, in accordance with the present invention.

FIG. 7 shows a live-cell microscopy image illustrating that T-DM1inhibits MK differentiation and disrupts proPLT formation by inducingabnormal tubulin organization, accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Blood platelets (PLTs) play a critical role in stimulating clotformation and repair of vascular injury. Morbidity and mortality frombleeding due to low PLT count is a major clinical problem encounteredacross a number of conditions including chemotherapy or radiationtreatment, trauma, immune thrombocytopenic purpura (ITP), organtransplant surgery, severe burns, sepsis, and genetic disorders. Despiteserious clinical concerns owing to their immunogenicity and associatedrisks, along with inventory shortages owing to high demand and shortshelf life, PLT transfusions total more than 10 million units per yearin the United States.

PLT production involves the differentiation of megakaryocyte (MKs),which sit outside sinusoidal blood vessels in the bone marrow (BM) andextend long, branching cellular structures (designated proPLTs) into thecirculation. ProPLTs experience vascular shear and function as theassembly lines for PLT production, containing PLT-sized swellings intandem arrays that are connected by thin cytoplasmic bridges. Althoughdetailed characterization of proPLTs remains incomplete, thesestructures have been recognized both in vitro and in vivo andproPLT-producing MKs in culture yield PLTs that are structurally andfunctionally similar to blood PLTs. PLTs are released sequentially fromproPLT tips. This mechanism is highly dependent on a complex network oftubulin and actin filaments that function as the molecular struts andgirders of the cell. Microtubule (MT) bundles run parallel to proPLTshafts, and proPLT longation is driven by MTs sliding over one another.During proPLT maturation, organelles and secretory granules trafficdistally over MT rails to become trapped at proPLT tips. Actin promotesproPLT branching and amplification of PLT ends. Live cell microscopy ofmurine MKs has been vital to this understanding, however most studies todate have been done in vitro on static MK cultures.

Thrombopoietin (TPO) has been identified as the major regulator of MKdifferentiation, and it has been used to produce enriched populations ofMKs in culture. In one reference, it was demonstrated that human PLTsgenerated in vitro from proPLT-producing MKs were functional. Sincethen, MKs have been differentiated from multiple sources, includingfetal liver cells (FLCs), cord blood stem cells (CBSCs), embryonic stemcells (ESCs), and induced pluripotent stem cells (iPSCs). However,current 2-D and liquid MK cultures fall orders of magnitude short of theestimated ˜2000 PLTs generated per MK in vivo. More recently, modular3-D knitted polyester scaffolds have been applied under continuous fluidflow to produce up to 6×10⁶ PLTs/day from 1 million CD34⁺ human cordblood cells in culture. While suggestive that clinically useful PLTnumbers may be attained, those 3-D perfusion bioreactors do notaccurately reproduce the complex structure and fluid characteristics ofthe BM microenvironment, and their closed modular design preventsvisualization of proPLT production, offering little insight into themechanism of PLT release. Alternatively, 3-D polydimethylsiloxane (PDMS)biochips adjacent ECM-coated silk-based tubes have been proposed toreproduce BM sinusoids and study MK differentiation and PLT productionin vitro. Although such devices recapitulate MK migration duringmaturation, they are not amenable to high resolution live-cellmicroscopy, and fail to reproduce endothelial cell contacts necessary todrive MK differentiation.

While MK differentiation has been studied in culture, the conditionsthat stimulate proPLT production remain poorly understood, particularlyin vivo. MKs are found in BM niches, and some evidence suggests thatcell-cell, cell-matrix, and soluble factor interactions of the BM stromacontribute to proPLT formation and PLT release. Indeed, the chemokineSDF-1 and growth factor FGF-4 recruit MKs to sinusoid endothelial cells.Extracellular matrix (ECM) proteins are another major constituent of theBM vascular niche, and evidence suggests that signaling throughtrans-membrane glycoprotein (GP) receptors regulate proPLT formation,PLT number and size (defects seen in e.g. Bernard-Soulier syndrome,Glanzmann's thrombasthenia). Collagen IV and vitronectin promote proPLTproduction, which can be inhibited by antibodies directed against theirconjugate integrin receptor, GPIbα. Likewise, fibrinogen regulatesproPLT formation and PLT release through GPIIbIIIa. While these findingsshed light on the environmental determinants of proPLT production, theyare limited by a reductionist approach. Therefore, new models thatincorporate the defining attributes of BM stroma complexity arenecessary to elucidate the physiological regulation of MKs into PLTs.

In the BM, proPLTs experience wall shear rates ranging from, 100 to10,000 s⁻¹ or, more particularly, from 500 to 2500 s⁻¹. While the roleof continuous blood flow on PLT thrombus formation has been studied,surprisingly little attention has been paid to the mechanism by whichshear forces regulate PLT release. When investigated, experiments havenot been physiologically representative. Some preliminary studies haveperfused MKs over ECM-coated glass slides, which select forimmobilized/adhered MKs without discriminating ECM-contact activationfrom shear. Alternatively, released proPLTs have been centripetallyagitated in an incubator shaker, which does not recapitulate circulatorylaminar shear flow, does not provide precise control of vascular shearrates, and is not amenable to high-resolution live-cell microscopy.Despite these major limitations, exposure of MKs to high shear ratesappears to accelerate proPLT production and while proPLTs cultured inthe absence of shear release fewer PLTs than those maintained at fluidshear stresses.

Microfluidic devices provide excellent platforms to generate andprecisely tune dynamic fluid flows, and thus mimic blood vesselconditions to deliver chemical cues to cells. Embedding microfluidicnetworks within cell-laden hydrogels has been shown to support efficientconvective transport of soluble factors through 3D scaffolds. Viable 3Dtissue contacts have been produced consisting of hepatocytesencapsulated in agarose, calcium alginate hydrogels seeded with primarychondrocytes, and endothelial cells embedded in 3D tubular poly(ethyleneglycol) hydrogels. Accordingly, the technology has been applied to thedevelopment of organs-on-a-chip, including liver, kidney, intestine, andlung. In addition, recent development of microvasculature-on-a-chipmodels have been used to study cardiovascular biology andpathophysiology in vitro. These studies emphasize the importance ofmimicking the physical microenvironment and natural chemical cues ofliving organs to study cellular and physiological development. Forexample, this is particularly important for drug-mediated inhibition ofPLT production. Since proPLT-producing MKs sit just outside bloodvessels in the BM, interacting with both the semi-solid ECMmicroenvironment of BM and fluid microenvironment of the circulation,biomimetic microfluidic biochips may achieve a model system toelucidating the relevant physiological mechanisms, such as thoseresponsible for drug-induced thrombocytopenia.

Turning now to FIG. 1, a schematic is shown illustrating an example of abiomimetic system 100 in accordance with various embodiments of thepresent invention. The system 100 includes a substrate 101, a firstchannel 102 and a second channel 104, wherein each channel is configuredto carry a flow of any fluid medium transporting or consisting of butnot limited to, for example, particles, cells, substances, particulates,materials, compositions and the like. In one embodiment, the system 100and/or substrate 101 may be constructed using cell-inert silicon-basedorganic polymers, such as polydimethylsiloxane (PDMS).

The first channel 102 includes a first channel input 106 and firstchannel output 108. Similarly, the second channel 104 includes a secondchannel input 110 and second channel output 112. The first channel 102and second channel 104 extend along a substantially longitudinaldirection, and are longitudinally and transversally dimensioned, as willbe explained, to achieve desired flow profiles, velocities, or rates,such as those present in a physiological system. In one aspect, the sizeof the longitudinal 130 and transverse 132 dimensions describing thechannels may be in a range consistent with an anatomical orphysiological structure, assembly, configuration or arrangement, such asin the case of bone marrow and blood vessels. By way of example, thelongitudinal 130 dimension may be in the range of 1000 to 30,000micrometers or, more particularly, in the range of 1000 to 3000micrometers, and the transverse 132 dimension may be in the range of 100to 3,000 micrometers or, more particularly, in the range of 100 to 300micrometers, although other values are possible. In another aspect, eachchannel may be prepared, conditioned, or manufactured to receive,localize, trap, or accumulate for example, particles, cells, substances,particulates, materials, compositions, and the like, from a traversingfluid medium.

The system 100 also includes a series of columns 114 that separate thefirst channel 102 and second channel 104. The long axes of the columns114 are generally arranged parallel to the longitudinal 130 dimension ofthe channels, the series of columns 114 extending for a distancesubstantially equal to the longitudinal 130 dimension of the channels.The columns 114 are separated by gaps, creating a series of gaps that,as illustrated, may be microchannels 116 that extend from the firstchannel 102 to the second channel 104 to create a partial fluidcommunication path passing between the columns 114. However, the term“microchannel” when referring to the gaps does not connote a particularwidth. For example, the gaps may be substantially greater or smallerthan the micrometer range. In one embodiment, the columns 114 andmicrochannels 116 are dimensioned such that particles, cells,substances, particulates, materials, compositions, and the like, maybind, adhere to or otherwise be confined to an area generally in thevicinity of the columns 114 and microchannels 116 and, thereby,harvested from an area proximate to the microchannels 116. As anexample, the longitudinal 130 and transverse 132 dimensions of thecolumns 114 may be in the range of 1 to 200 micrometers, while thelongitudinal 130 dimension of the microchannels 116, defined by theseparation distances or gaps between the columns 114, may be in therange of 0.1 to 20 micrometers, although other values are possible.

Flow in the first channel 102 is established through a first source orinput 118 configured for deliver a first medium, and a first outlet 120,configured for extracting the first fluid medium. Similarly, flow of asecond fluid medium in the second channel 104 is established through asecond source or inlet 122 to a second outlet 124. The first input orsource 118 and the second source of inlet 122 may be arranged to includea pump or other system for delivering a controlled flow. In oneconfiguration, the first outlet 120 or second outlet 124 may also befitted with or followed by elements, components, devices or systemsdesigned to capture, store and/or separate a desired material orsubstance from a first or second fluid medium, such as for example,human blood platelets, or thrombocytes. That is, flow velocities or flowrates of the first fluid medium between the first channel input 106 andfirst channel output 108, and of the second fluid medium between thesecond channel input 110 and second channel output 112, may beestablished by way of fluid communication of system 100 with any numberof sources, such as microfluidic pumps and drains, and may be sustainedfor any desired or required amount of time. Control and manipulation offlow may be realized by integrating elements, such valves, sources anddrains, with the system 100, or may be achieved by external interfacingor coupling of the system 100 with various components for fluidactuation and flow regulation.

As will be described, flow velocities or rates in the first channel 102may be configured to be substantially different from flow velocities orrates in the second channel 104, as desired, or as required forrecapitulating, modeling, or duplicating physiological elements,constituents and conditions such as, for example, those found in bonemarrow and blood vessels. In another embodiment, flow velocities orrates may be controlled in a manner that duplicates physiological shearrates and profiles, such as vascular shear rates and profiles.

The system 100 may also include filtration elements 126, which may takeany shape or form, arranged along the paths of each of the first andsecond fluid mediums and designed to capture or remove from thetraversing fluid mediums any kind of debris, dust and any othercontaminants or undesirable materials, elements, or particulates. In oneconfiguration, filtration elements 126 are situated in proximity to thefirst inlet 118 and second inlet 122. The system 100 further includesflow resistive elements 128, which may take a variety of shapes orforms, arranged along the paths of each of the first and second fluidmediums and designed to control flow forces or damp fluctuations in flowrate. In one configuration, flow resistive elements 128 may be situatedfollowing each of the filtration elements 126 along the paths of each ofthe first and second fluid mediums.

In one configuration, recreating human bone marrow (BM) vascular nicheex vivo may be achieved by selectively filling the first channel 102with bone powder, proteins, such as CI, CIV, FG, FN, VN, LN and VWF,gels such as agarose, alginate, and matrigel or solutions such as PBS,HBS, DMEM EGM or other media, alone and in combination. Alternatively,ECM proteins may be patterned directly onto glass surfaces prior toadhesion of biochips to surface slides using proteinmicro/nano-stamping, or following microfluidic device assembly usingparallel microfluidic streams. Local component concentration may beadjusted by regulating microfluidic stream flow rate during infusion,with focus on alignment and 3-D arrangement.

In another configuration, recapitulating human BM vasculature may beachieved by selectively coating the second channel 104 by culturing withendothelial cells at 37 degrees C. and 5 percent CO₂. Endothelial cellsmay be fixed with 4% formaldehyde, and probed for cellular biomarkers toresolve cellular localization and architecture. The second channel 104of endothelialized BM biochips may be perfused with a fluorescent orcolorimetric medium such as FITC-dextran or with beads, and visualizedby live-cell microscopy to assess sample/cell/molecule diffusion anddetermine vascular permeability.

The system 100 in accordance with the present invention can provide aplatform for recapitulating physiological conditions, such as those ofhuman BM, by replicating the dimensions, environments and conditionsfound in human venules using a biomimetic microfluidic device. Themicrofluidic channels separated by columns spaced closely apartexperiencing controlled environments and flow conditions represent arealistic physiological model that may be employed to produce functionalPLTs. In this manner, MK trapping, BM stiffness, ECM composition,micro-channel size, hemodynamic vascular shear, and endothelial cellcontacts may be tailored to reproduce human BM in vitro.

Specific examples of materials and methods utilized in this approach aredetailed below. It will be appreciated that the examples are offered forillustrative purposes only, and are not intended to limit the scope ofthe present invention in any way. Indeed, various modifications of theinvention in addition to those shown and described herein such as theapplication of this invention to model the blood brain barrier or studymolecular diffusion across separate mediums will become apparent tothose skilled in the art from the foregoing description and thefollowing examples and fall within the scope of the appended claims. Forexample, specific dimensions, configurations, materials, cell types,particulates, flow medium and flow rates, fabrication methods andrecipes, as well as imaging, processing and analysis methods, and so on,are provided, although it will be appreciated that others may also beused.

EXAMPLES Microfluidic Device Design and Fabrication

As shown in FIG. 1, microfluidic devices were fabricated using softlithography, consisting of two channels containing passive filters, fortrapping air bubbles and dust, followed by fluid resistors, used to dampfluctuations in flow rate arising during operation. The channels mergeto a rectangular area 1300 micrometers long, 130 micrometers wide, and30 micrometers deep, separated by a series of columns (10 micrometerswide and 90 micrometers long) spaced 3 micrometers apart. To ensureefficient gas exchange and support high-resolution live-cell microscopyduring cell culture, microfluidic devices were constructed from acell-inert silicon-based organic polymer bonded to glass slides.

AutoDesk software in AutoCAD was used to design the desired 2D patternand printed on a photolithography chrome mask. Silicon wafers(University Wafers, Boston, Mass.) were spin coated with SU-8 3025photoresist (Michrochem, Newton, Mass.) to a 30 micrometers filmthickness (Laurell Technologies, North Wales, Pa.), baked at 65 degreesC. for 1 minute and 95 degrees C. for 5 minutes, and exposed to UV light(˜10 mJ cm⁻²) through the chrome mask for 30 seconds. The unbound SU-8photoresist was removed by submerging the substrate into propyleneglycol monomethyl ether acetate for 7 minutes. Polydimethylsiloxane(PDMS) was poured onto the patterned side of the silicon wafer,degassed, and cross-linked at 65 degrees C. for ˜12 hours. After curing,the PDMS layer was peeled off the mold and the inlet and outlet holeswere punched with a 0.75 mm diameter biopsy punch. The channels weresealed by bonding the PDMS slab to a glass cover slide (#1.5, 0.17×22×50mm, Dow Corning, Seneffe, Belgium) following treatment with oxygenplasma (PlasmaPrep 2, GaLa Instrumente GmbH, Bad Schwalbach, Germany).Samples were infused into the microfluidic device via PE/2 tubing(Scientific Commodities, Lake Havasu City, Ariz.) using 1 mL syringesequipped with 27-gauge needles (Beckton Dickinson, Franklin Lakes,N.J.). Flow rates of liquids were controlled by syringe pumps (PHD 2000,Harvard Apparatus, Holliston, Mass.).

Microfluidic Device Operation

Devices were coated with a 0.22 μm filtered 10% BSA solution (Millipore,Billerica, Mass.) for 30 minutes to prevent direct cell contact withglass. Primary MKs and media were infused in the first inlet 118 andsecond inlet 122, respectively, at a rate of 12.5 μL/hour using atwo-syringe microfluidic pump (Harvard Apparatus, Holliston, MA). Whenthe first outlet 120 is closed, both input solutions are redirectedtoward the second outlet 124 causing primary MKs to trap.

Extracellular Matrix Composition Modeling (2D)

Microfluidic devices were selectively coated with extracellular matrixproteins by perfusing the channels with rhodamine-conjugated fibrinogen(1 mg/mL) or fibronectin (50 μg/mL, Cytoskeleon Inc., Denver, Colo.) for30 minutes. Samples were perfused in parallel through both inlets andcollected through both outlets so that laminar flow streams did not mix.Devices were washed with lx PBS and coated with 0.22 μm filtered(Millipore, Billerica, Mass.) 10% BSA solution (Roche, South SanFrancisco, Calif.) for 30 minutes to coat any remaining exposed glass.

BM Stiffness Modeling (3D)

Primary MKs were re-suspended in 1% sterile alginate with an averagemolecular weight of 150-250 kD (Pronova SLG100, FMC biopolymer, Norway)in culture media and perfused across the microfluidic device (firstinlet 118, second outlet 124) until MKs became trapped. The secondchannel was then selectively perfused with 1×PBS to remove alginate fromthis channel. To make a homogenous alginate gel, 30 mM nanoparticlecalcium carbonate (mkNANO, Canada) was used as a calcium source anddissolved in 60 mM slowly hydrolyzing D-Glucono-δ-lactone(Sigma-Aldrich, St. Louis, Mo.), which releases the calcium in thesolution (in review Khavari et al NJP 2013). The calcium carbonatesuspension was perfused along the second channel until the alginatesolution retained in the first channel became polymerized (-20 minutes).The second channel was then selectively washed with 1× PBS and replacedwith culture media. To determine the alginate gel's mechanicalproperties 0.25 percent, 0.5 percent, 1.0 percent, and 2.0 percentalginate gels were prepared and their frequency-dependent shear moduliwere measured by rheology at 37° C. (Ares G2 TA instruments, New Castle,Del.).

Sinusoidal Blood Vessel Contact Modeling (3D)

Microfluidic devices were selectively coated with 50 μg/mL fibronectin(Cytoskeleon Inc., Denver Colo.) and 10 percent BSA (Roche, South SanFrancisco, Calif.), as described above, and transferred to a 37 degreesC., 5 percent CO2 incubator. 10,000,000 HUVECs/mL in EBM media (Lonza,Basel, Switzerland) were seeded over the fibronectin-coated channel at12.5 uL/hour and permitted to adhere to this surface over a period of 3hours. The inlet sample was replaced with cell-free EBM media andperfused through the channel until HUVECs reached confluency (2-8 days).Cells were stained with 5 μM CellTracker Red and 1 μg/mL Hoescht 33342(Invitrogen, Carlsbad, Calif.) for 45 minutes, washed in fresh media orfixed in 4% formaldehyde and visualized by confocal-fluorescencemicroscopy.

Vascular Shear Rate Modeling (3D)

The shear stresses imparted on the MKs were estimated with acomputational model of the fluid dynamics within the microfluidicdevice. A commercial finite element method software (COMSOL) was used tosolve the Navier-Stokes equation. The steady-state Navier Stokes flowequation for incompressible flow is:

ρ(

·

)=−

p+μ

²

+f   (1)

where ρ is the fluid density,

is the flow velocity, ρ is the pressure, μ is the fluid viscosity and fis the body forces action on a fluid. Equation (1) was solved in a threedimensional computational domain replicating the exact dimensions of themicrofluidic device. It was assumed that the fluid within the device hada viscosity and density of water (0.001 Pa s and 1000 kg/m3,respectively). No slip boundary conditions were assumed at the walls ofthe channels. The infusion flow rates ranged from 12.5-200 μL/hr. Atriangular mesh, which was made finer at the slits, was used todiscretize the domain. The model contained 315,317 degrees of freedom.Mesh independence, as was confirmed by obtaining less than a 10 percentdifference between shear rates, was found between 251,101 and 415,309degrees of freedom. The steady state solutions were obtained using theUMFPACK linear system solver.

Primary Mouse Megakaryocyte Culture

Mouse FLCs were collected from WT CD1 mice (Charles River Laboratories,Wilmington, Mass.) and MKs were cultured.

Electron Microscopy

Megakaryocyte input and bioreactor effluent were fixed with 1.25 percentparaformaldehyde, 0.03 percent picric acid, 2.5 percent glutaraldehydein 0.1-M cacodylate buffer (pH 7.4) for 1 h, post-fixed with 1% osmiumtetroxide, dehydrated through a series of alcohols, infiltrated withpropylene oxide, and embedded in epoxy resin. Ultrathin sections werestained and examined with a Tecnai G2 Spirit BioTwin electron microscope(Hillsboro, Oreg.) at an accelerating voltage of 80 kV. Images wererecorded with an Advanced Microscopy Techniques (AMT) 2-K chargedcoupled device camera, using AMT digital acquisition and analysissoftware (Advanced Microscopy Techniques, Danvers, Mass.).

Immunofluorescence Microscopy

Megakaryocytes, released proPLTs, or bioreactor effluent were purifiedand probed. Samples were either incubated with 5 μM CellTracker Green(Invitrogen, Carlsbad, Calif.) for 45 minutes, washed in fresh media andvisualized by live-cell fluorescence microscopy, or fixed in 4%formaldehyde and centrifuged onto poly-L-lysine (1 μg/mL)-coatedcoverslides. For analysis of cytoskeletal components, samples werepermeabilized with 0.5 percent Triton-X-100, and blocked inimmunofluorescence blocking buffer (0.5 g BSA, 0.25 ml 10% sodium azide,5 ml FCS, in 50 ml 1×PBS) overnight before antibody labeling (55). Todelineate the microtubule cytoskeleton, samples were incubated with arabbit polyclonal primary antibody for mouse or human β1-tubulin. Todelineate the actin cytoskeleton, samples were incubated with Alexa 568phalloidin (Invitrogen, Carlsbad, Calif.). Cell nuclei were labeled with1 μg/mL Hoescht 33342 (Invitrogen, Carlsbad, Calif.). To correct forbackground fluorescence and nonspecific antibody labeling, slides wereincubated with the secondary antibody alone, and all images wereadjusted accordingly. Samples were examined with a Zeiss Axiovert 200(Carl Zeiss, Thornwood, N.Y.) equipped with 10× (numerical aperature,0.30) Plan-Neofluar air and 63× (numerical aperature, 1.4)Plan-ApoChromat oil immersion objectives, and images were obtained usinga CCD camera (Hamamatsu Photonics, Boston, Mass.). Images were analysedusing the Metamorph version 7.7.2.0 image analysis software (MolecularDevices, Sunnyvale, Calif., USA) and ImageJ version 1.47p software (NIH,http://rsb.info.nih.gov.ezp-prod1.hul.harvard.edu/ij/).

Cell Size and Morphology Determination

Cells were individually thresholded and high-content cytoplasmic areaand perimeter measurements were performed in ImageJ usinginvestigator-coded software, outlined below. Analysis was confirmed bymanual inspection of all samples, and improperly thresholded cells wereexcluded from the analysis. MK diameters were calculated from areameasurements to account for non-circular cells. More than 2000 cellswere counted for each condition, and analysis of MK area and effluentcomposition was performed for at least three independent samples.Statistical significance was established using a 2-tailed Student t testfor paired samples. Error bars represent one standard deviation aboutthe mean.

Live Cell Microscopy

For shear cultures, MKs were loaded onto ‘naked’ microfluidic devices(only BSA-coated), and the infusion rate was doubled incrementally from12.5 μL/hr to 200 μL/hr over a 2 hour period. For static cultures,isolated MKs were pipetted into chambers formed by mounting a glasscoverslide coated with 3% BSA onto a 10 mm petri dish with a 1 cm holeand cultured for 24 hours. Both static and shear cultures weremaintained at 37 degrees C. and 5 percent CO2 and examined on a ZeissAxiovert 200 (Carl Zeiss, Thornwood, N.Y.) equipped with 10× (numericalaperature, 0.30) Plan-Neofluar air objective. Differential interferencecontrast (DIC) images were obtained using CCD camera (HamamatsuPhotonics, Boston, Mass.) at either 2 second (shear cultures) or 20minute (static cultures) intervals. Images were analyzed using theMetamorph version 7.7.2.0 image analysis software (Molecular Devices,Sunnyvale, Calif., USA) and ImageJ software version 1.47p. ProPLTextension rates were determined manually for over 200 MKs from at leastthree independent samples. For PLT spreading experiments, effluent wascollected from microfluidic devices after 2 hours and pipetted intouncoated static culture chambers, described above. PLTs were permittedto contact glass by gravity sedimentation and spreading was captured at5 second intervals over a 5 minute period.

GFP-β1 Tubulin Retro Viral Transfection

Dendra2-fused β1 tubulin was cloned into pMSCV plasmids. HEK 293 cellspackaging cells were cultured in DMEM supplemented with 10% fetal bovineserum (FBS) to 30-50 percent confluency. Transfection of HEK 293 cellswas performed using 2 μg of DNA plasmids encoding gag/pol, vsvG, and theβ1 tubulin fused with Dendra2 in the pMSCV vector. After medium exchangethe following day, cells were incubated for 72 hours for virusproduction. The supernatant was filtered through a 0.22 μm filter(Millipore, Billerica, Mass.), and aliquots were stored at −80 degreesC. On the second day of culture, MKs isolated from fetal liver culturesdescribed above were resuspended in DMEM containing 10 percent FBS, 8μg/mL polybrene (Sigma), and the retroviral supernatant. Samples weretransferred to a 6-well plate, centrifuged at 800×g for 90 minutes at 25degrees C. and then incubated at 37 degrees C. for 90 minutes. Followingincubation, MKs were washed by centrifugation and resuspended in freshDMEM containing 10 percent FBS and TPO. MKs were allowed to mature untilday 4 of culture and then isolated by a BSA gradient, as previouslydescribed.

Flow Cytometry

Platelets were collected from the released proPLT fraction of static MKcultures or bioreactor effluent and examined under resting conditions.Samples were probed with FITC-conjugated antibodies against CD42a orCD41/61 (Emfret Analytics, Eibelstadt, Germany) and run on a FACSCaliburflow cytometer (Beckton Dickinson). PLTs were gated by theircharacteristic forward- and side-scattering as they passed through thedetector, and their total fluorescence intensity was calculated aftersubtraction of a FITC-conjugated IgG antibody specificity control(Emfret Analytics). Quantization of PLT yield was determined by dividingnet GP IX+ PLT production by net GP IX+ MK depletion over effluentcollection period, and was performed for at least 3 independent samplesResults were identical for GP IIbIIIa+ cells.

Image Analysis

The digital images acquired in Metamorph were analyzed using ImageJ andAdobe Photoshop CS3 (Adobe Systems, San Jose, Calif.). Dividing linesexplicitly separate different images, or separate regions of the sameimage. No specific features within an image were enhanced, obscured,moved, removed, or introduced, and adjustments made to the brightness,contrast, and color balance were linearly applied to the whole image.

Microfluidic Device Models Physiological Characteristics of Human BM

To recapitulate physiological conditions, each of the channels wereselectively coated with fibrinogen and fibronectin, respectively toreproduce ECM composition of the BM and blood vessel microenvironments(shown in FIG. 2A). By running flow across the microfluidic device,primary MKs infused along a first channel would become sequentiallytrapped between the columns and extend proPLTs into the second channel(shown in FIG. 2B), recapitulating physiological proPLT extension. Tomodel 3D ECM organization and physiological BM stiffness (250 Pa), MKswere infused in a 1 percent alginate solution that was polymerizedwithin the microfluidic device, selectively embedding the MKs inalginate gel within the first channel while retaining vascular flow inthe second channel. Alginate did not inhibit proPLT production, and MKdistance from the second channel could be controlled.

Human umbilical vein endothelial cells (HUVECs) were selectively seededalong the second channel, and grown to confluency to produce afunctional blood vessel (shown in FIG. 2C). In addition, MK behavior wasmonitored by 10×-150× magnification, high-resolution live-cellmicroscopy, and the released PLTs were collected from the effluent. FIG.2D shows the complete system illustrating operation. Laminar fluid shearrates were characterized (shown in FIG. 2E), and were tightly controlledusing two microfluidic pumps (one for the first channel and one for thesecond channel). Shear rates within the device were linearlyproportional to infusion rates and were adjusted to span thephysiological range (500-2500 s⁻¹). While shear rates at emptymicrochannel junctions increased with distance from the first channel(shown in FIG. 2F), upon a MK trapping, flow was redirected to the nextavailable gap such that MKs continued to experience physiological(between 760 and 780 s⁻¹) shear rates at these sites (shown in FIG. 2G).

Vascular Shear Triggers proPLT Production, Physiological Extension, andRelease

In vivo BM MKs extend proPLTs in the direction of blood flow and releasePLTs, proPLTs, large cytoplasmic fragments (prePLTs), and even whole MKsinto sinusoidal blood vessels which may be trapping in the pulmonarymicrovascular bed, or otherwise maturing in the circulation. Todetermine the effect of physiological shear on PLT production, mousefetal liver culture-derived (mFLC) MKs were isolated on culture day 4and characterized by size and ploidy before being infused into themicrofluidic device (shown in FIG. 3A).

One of the major challenges in producing transfuseable PLTs in vitro hasbeen identifying factors that trigger proPLT production. Under staticconditions MKs begin producing proPLTs −6 hours post-isolation, andreach maximal proPLT production at 18 hours (shown in FIG. 3B). Bycomparison, MKs under physiological shear (shown in FIG. 3C at roughly500 s⁻¹) began producing proPLTs within seconds of trapping, reachingmaximal proPLT production and biochip saturation within the first 2hours of culture. MKs cultured under physiological shear produced fewer,longer proPLTs that were less highly branched relative to staticcultures. ProPLTs in shear cultures were uniformly extended into thelower channel and aligned in the direction of flow against the vascularchannel wall, recapitulating physiological proPLT production. Thepercent of proPLT-producing MKs under physiological shear were doubledover static cultures to roughly 90% (shown in FIG. 3D).

Another major challenge in generating clinical numbers of PLTs forinfusion has been that in vitro cultures extend proPLTs at asignificantly slower rate than what has been observed in vivo.Application of physiological shear in our microfluidic device increasedproPLT extension rate by an order of magnitude above static culturecontrols to roughly 30 μm/min (shown in FIG. 3E), which agrees withphysiological estimates of proPLT extension rate from intravitalmicroscopy studies in living mice and support increased PLT productionin vitro.

Early histological studies in both humans and mice have predicted thatwhole MKs, as well as MK fragments may be squeezing through gaps orfenestrations in the vascular endothelium lining BM blood vessels totrap in the pulmonary circulatory bed. Large PLT intermediates calledprePLTs were recently discovered in blood, and venous infusion of mBM-and FLC-derived MKs and prePLTs into mice produced PLTs in vivo. In thepresent study, 100 μm+ diameter MKs were routinely observed squeezingthrough 3 μm (shown in FIG. 4A) and 1.5 μm gaps, or extending large MKfragments (shown in FIG. 4B), supporting a model of vascular PLTproduction. In addition, abscission events were routinely captured byhigh-resolution live-cell microscopy and occurred at variable positionsalong the proPLT shaft, releasing both prePLT-sized intermediates (3-10μm diameter) and PLTs (1.5-3 μm diameter) (shown in FIG. 4C and FIG.4D). Following each abscission, the resulting proPLT end formed a newPLT-sized swelling at the tip, which was subsequently extended andreleased, repeating the cycle (shown in FIG. 4E).

While shear rates were kept constant, proPLT extension rates varied atdifferent positions along the shaft, predictive of a regulatedcytoskeletal driven mechanism of proPLT elongation (shown in FIG. 4C).Increasing microfluidic shear rates within the physiological range didnot affect the median proPLT extension rate or the distribution ofproPLT extension rates in culture (shown in FIG. 4F), and proPLTprojections in MKs retrovirally transfected to express GFP-β1 werecomprised of peripheral microtubules (MTs) that formed coils at thePLT-sized ends (shown in FIG. 4G). ProPLTs reached lengths exceeding 5mm, and resisted shear rates up to 1000 s⁻¹ in vitro; recapitulatingphysiological examples of proPLT production from intravital microscopy,and demonstrating that abcission events were not caused by shear. Toconfirm that shear-induced proPLT extension was cytoskeletal-driven, MKswere incubated with 5 μM Jasplankinolide (Jas, actin stabilizer) or 1 mMerythro-9-(3-[2-hydroxynonyl] (EHNA, cytoplasmic dynein inhibitor) priorto infusion in microfluidic device. Both Jas and EHNA inhibitedshear-induced proPLT production (shown in FIG. 4H and FIG. 4I) and PLTrelease under both static and physiological shear conditions.

Derived PLTs Manifest Structural and Functional Properties of Blood PLTs

PLTs are anucleate discoid cells ˜1-3 μm in diameter that expressbiomarkers GP IX and IIbIIIa on their surface, and are characterized bya cortical MT coil of 6-8 MTs encircling an actin-based cytoskeletalnetwork. To establish PLT yield, biomarker expression, and forward/sidescatter and relative concentration of glycoprotein (GP) IX+ mFLC-MKswere measured by flow cytometry immediately before infusion in ourmicrofluidic device on culture day 4 (shown in FIG. 5A). Effluent wascollected 2 hours post infusion and compared to mFLC-MK input (shown inFIG. 5B). Input MKs and effluent PLTs both expressed GP IX and IIbIIIaon their surface, and displayed characteristic forward/side scatter. Theapplication of shear shifted the cellular composition of the effluenttoward more PLT-sized GPIX+ cells relative to static culture supernatantisolated on culture day 5 (shown in FIG. 5C). 85±1% of MKs wereconverted into PLTs over 2 hours, which agreed with our quantitation ofpercent proPLT production (FIG. 3D) and constitutes a significantimprovement over static cultures (FIG. 5D). Continuous perfusion ofroughly 500 s⁻¹ shear over 2 hours in our microfluidic device yieldedroughly 21 PLTs per MK and constitutes a major advance in PLT productionrate over existing culture approaches that generate comparable PLTnumbers over a much longer period of time (6-8 days).

To quantify the morphological composition of our product, the effluentfrom our microfluidic device was probed for β1 tubulin (PLT-specifictubulin isoform) and Hoescht (nuclear dye), and analyzed byimmunofluorescence microscopy. Cells were binned according to theirmorphology and size, and compared to static MK culture supernatants. Theapplication of shear shifted the cellular composition of the effluenttoward more PLT-sized β1 tubulin+Hoescht-cells (shown in FIG. 5E), whichagreed with flow cytometry data (FIG. 5C) and resulted in a product thatwas more similar in composition to the distribution of PLT intermediatesin whole blood. Quantitation of free nuclei in effluent confirmedincreased microfluidic device-mediated PLT production relative to staticcultures and established PLT yields of roughly 20±12 PLTs per MK, whichagree with flow cytometry data.

Resting PLTs contain characteristic invaginations of the surfacemembrane that form the open canalicular system, a closed channel networkof residual endoplasmic reticulum that form the dense tubular system,organelles, specialized secretory granules, and will flatten/spread oncontact activation with glass. Microfluidic device-generated PLTs wereultrastructurally indistinguishable from mouse blood PLTs bythin-section transmission electron; and contained a cortical MT coil,open canalicular system, dense tubular system, mitochondria, alpha- anddense-granules (as shown in FIG. 5F). Microfluidic device-generated PLTsand PLT intermediates displayed comparable MT and actin organization tomouse blood PLTs by immunofluorescence microscopy (as shown in FIG. 5G),and spread normally on contact-activation with glass, forming bothfilpodia and lamellipodia.

Application of the Microfluidic Device to Human PLT Production

To generate human PLTs, mFLC-MK in our microfluidic device were replacedwith hiPSC-derived MK, which provide a virtually unlimited source of MKsfor infusion. hiPSC-MKs were isolated on culture day 15, once they hadreached maximal diameter of 20-60 μm (shown in FIG. 6A), and wereultrastructurally similar to primary human MKs (shown in FIG. 6B). Instatic culture, hiPSC-MKs began producing proPLTs at 6 hourspost-isolation, and reached maximal proPLT production at 18 hours (shownin FIG. 6C). By comparison, hiPSC-MKs under physiological shear (about500 s⁻¹) began producing proPLTs immediately upon trapping, andextended/released proPLTs within the first 2 hours of culture (shown inFIG. 6D). The percent proPLT-producing hiPSC-MKs under shear wereincreased significantly over static cultures (˜10%) to roughly 90% (asshown in FIG. 6E).

ProPLT extension rates were slightly lower than mFLC-MK controls (˜19μm/min versus 30 μm/min) (shown in FIG. 6F) and more closelyapproximated physiological controls. Microfluidic device-generated PLTsdisplayed forward and side scatter, and surface biomarker expressioncharacteristic of human blood PLTs, were ultrastructurallyindistinguishable from human blood PLTs by thin-section transmissionelectron (shown in FIG. 6G), displaying comparable MT and actinexpression to human blood PLTs by immunofluorescence microscopy (shownin FIG. 6H), spreading normally on contact-activation with glass, andforming both filpodia and lamellipodia (shown in FIG. 6I). Takentogether these data demonstrate that hiPSC-MKs can be applied to ourbiomimetic microfluidic device to generate potentially unlimited numbersof functional human PLTs.

Application of the Microfluidic Device to Drug Development

Thrombocytopenia may appear suddenly and often unintentionally,potentially causing major bleeding and death. Antibody and cell-mediatedautoimmune responses have been shown to cause thrombocytopenia. Inaddition, thrombocytopenia may also be triggered by a wide range ofmedications, including cancer drugs, such as dasatinib. Animal modelsare generally poor predictors of safety and efficacy of medications inhumans, and clinical studies are time-consuming, expensive, andpotentially harmful. Microfluidic devices designed to mimic human BMrepresent an area of innovation of major clinical importance, offeringan efficient and realistic platform to investigate the effects of avariety of medications upon BM and MK biology.

PLT survival and clearance rates are usually measured through infusionstudies using flow cytometry. Quantification of the rate and extent ofproPLT production, however, is not amenable to this approach, andrequires direct visualization to establish at what stagethrombocytopoiesis is affected. By contrast, the application ofmicrofluidic devices offers a great platform to study drug effects onPLT production, one that may facilitate the identification of newregulators of PLT production and elucidate the mechanism of clinicallysignificant drug-induced thrombocytopenias.

As proof of concept, high-content live-cell microscopy was employed toidentify the express GFP-PI tubulin (live-cell microscopy) mechanism bywhich trastuzumab emtansine (T-DM1), an antibodydrug conjugate currentlyin clinical development for breast cancer, affects PL T production.These studies revealed that T-DM1 inhibits MK differentiation, anddisrupts proPLT formation by inducing abnormal tubulin organization (asshown in FIG. 7). Defining the pathways by which therapeutics such asT-DM1 affect MK maturation and proPLT production may yield strategies tomanage drug-induced thrombocytopenias and regulate PLT production invivo.

The approach of the present invention capitalizes on a highly novelmicrofluidic design to recapitulate human BM and blood vessel physiologyex vivo, and generate an alternative source of functional human PLTs forinfusion. While clinically desirable to meet growing transfusion needsand obviate risks currently associated with platelet procurement andstorage, 2 major quantitative roadblocks have thus far persisted in thedevelopment of donor-independent PLTs for therapeutic use: (1)generating sufficient numbers (˜3×10⁸) of human MKs to support theproduction of one PLT transfusion unit (˜3×10¹¹ PLTs), and (2)generating physiological numbers of functional human PLTs (˜10³-10⁴) perMK. The development of human embryonic stem cell cultures (hESC), andmore recently, human induced pluripotent stem cell cultures (hiPSC),offer a potentially unlimited source of progenitor cells that can bedifferentiated into human MKs in vitro to address the first quantitativeroadblock. Indeed, because PLTs are anucleate, PLT microfluidicdevice-derived units could be irradiated prior to infusion, addressingconcerns that cellular products derived from hESC or hiPSCs could beoncogenic or teratogenic.

Attempts to study the environmental drivers of PLT production have beenconstrained by reductionist approaches, and a major limitation of 2Dliquid cultures has been their inability to account for 3D BMcomposition and stiffness, directionality of proPLT extension, andproximity to venous endothelium. Likewise, while proPLT-producing MKsentering sinusoidal blood vessels experience wall shear rates of 500 to2500 s⁻¹, attempts to model vascular flow by perfusing MKs overECM-coated glass slides have selected for immobilized/adhered MKs, andhave been unable to discriminate ECM-contact activation from shear.Alternatively, released proPLTs have been centripetally agitated in anincubator shaker, which does not recapitulate laminar shear flow in BMblood vessels, does not provide precise control of vascular shear rates,and is not amenable to high-resolution live-cell microscopy.Nonetheless, despite these limitations, exposure of MKs to high shearrates (1800 s⁻¹) accelerated proPLT production, and proPLTs cultured inthe absence of shear released significantly fewer PLTs than thosemaintained at fluid shear stresses of ˜0.5 Pa for 2 hours. Moreover,recent advances in multiphoton intravital microscopy have providedincreasing resolution of proPLT production in vivo and confirmed theimportance of vascular flow on proPLT extension and PLT release. Whilethese studies have provided physiologically accurate examples of in vivoproPLT production, poor resolution and limited control of themicroenvironment has prohibited detailed study of how the BMmicroenvironment contributes to PLT release.

Mounting evidence that cell-cell contacts, extracellular matrix (ECM)composition and stiffness, vascular shear rates, pO2/pH, soluble factorinteractions, and temperature contribute to proPLT formation and PLTrelease have suggested that recapitulating BM and blood vesselmicroenvironments within a 3D microfluidic culture system is necessaryto achieve clinically significant numbers of functional human PLTs.Indeed, modular 3D knitted polyester scaffolds have been applied undercontinuous fluid flow to produce up to 6×10⁶ PLTs/day from 1×10⁶ CD34+human cord blood cells in culture. While suggestive that clinicallyuseful numbers of culture-derived human PLTs are attainable, 3Dperfusion bioreactors have not accurately reproduced the complexstructure and fluid characteristics of the BM microenvironment, andtheir closed modular design has prevented direct visualization of proPLTproduction, offering little insight into the mechanism of PLT release.Alternatively, 3D PDMS biochips adjacent ECM-coated silk-based tubeshave been proposed to reproduce BM sinusoids and study MKdifferentiation and PLT production in vitro. While capable ofrecapitulating MK migration during maturation, this design is notamenable to high resolution live-cell microscopy, and does not reproduceendothelial cell contacts necessary to drive MK differentiation.

By comparison, the microfluidic device design of the present inventionoffers the complete package, allowing significant improvement in time toPLT release and an increased total PLT yield. Also, application ofvascular shear rates within the microfluidic device induces proPLTproduction, and reproduces physiological proPLT extension and release.Furthermore, MKs are capable of squeezing through small gaps to enterthe circulation and releasing prePLT intermediates under physiologicalflow conditions. The product resulting from continuous perfusion of MKsin the microfluidic device of the present invention approachedphysiological PLT concentratons, and manifested both structural andfunctional properties of blood PLTs. Finally, PLT microfluidic devicescould be applied to human MK cultures to produce functional human PLTs.Although PLT yield per MK fell short of theoretical estimates, theobservation that MK cultures routinely released large MK fragments(prePLTs, proPLTs) as well as MK themselves into the effluent channel,suggests that actual PLT numbers may depend on the furtherdifferentiation of PLT intermediates into PLTs in supportivemicroenvironments such as the lung or circulating blood. Indeed, whenmFLC-derived proPLTs were infused into mice, these were rapidlyconverted into PLTs over a period of 12-24 hours. Interestingly, whileCMFDA-labeled PLTs in this study were readily detected in the blood,larger prePLT intermediates were not, suggesting that they may betrapping in a microcirculation of the lung. Likewise, when mFLC andBM-derived MKs were infused into mice they almost exclusively localizedto the lungs and released PLTs within the first two hours. In bothcases, it is almost certain that vascular shear rates, soluble factorinteractions in the blood, and endothelial cell contacts regulate thisprocess, and examining how local microenvironments in these tissuescontribute to terminal PLT production warrant further investigation.

By combining the major elements of BM physiology including 3D ECMcomposition and stiffness, cell-cell contacts, vascular shear rates,pO2/pH, soluble factor interactions, and temperature within a singlemicrofluidic system, the approach of the present invention offersunprecedented control of ex vivo microenvironments and a biomimeticplatform for drug development. Moreover, support of high-resolutionlive-cell microscopy permits direct visualization of cells duringculture and provides a window into poorly characterized physiologicalprocesses. Lastly, the microfluidic device design can be easily scaledby mirroring effluent channels on either side of a central channel,elongating the device to support greater numbers of columns, andpositioning multiple units in parallel within a larger microfluidicdevice matrix. Continuous harvesting of hiPSC-MKs in longer devices mayresult in clinically significant numbers of PLTs to perform, forexample, traditional aggregometry tests of PLT function, and in vivoxeno-transfusion studies in immune-suppressed mice to measure increasesin PLT counts, which require roughly 10⁸ PLTs per study.

In summary, the present invention has demonstrated a system and methodto recapitulate human BM and sinusoidal blood vessel microenvironmentsfor generating human platelets in an approach amenable to highresolution imaging. The biomimetic microfluidic system may be fabricatedusing PDMS bonded to glass in a configuration that includes microfluidicchannels separated by a series of columns. The channels can beselectively coated with ECM and human endothelial cells to simulaterealistic physiological conditions. Round or proPLT-producing MKsinfused along one channel can sequentially become trapped between thecolumns, and extend platelet-producing proplatelets into the otherchannel. Stimulated by controllable physiological shear rates andregulated microenvironments, the released PLTs entering the fluid streamcan be collected from the effluent, and the process may be visualized byhigh-resolution live-cell microscopy.

The various configurations presented above are merely examples and arein no way meant to limit the scope of this disclosure. Variations of theconfigurations described herein will be apparent to persons of ordinaryskill in the art, such variations being within the intended scope of thepresent application. In particular, features from one or more of theabove-described configurations may be selected to create alternativeconfigurations comprised of a sub-combination of features that may notbe explicitly described above. In addition, features from one or more ofthe above-described configurations may be selected and combined tocreate alternative configurations comprised of a combination of featureswhich may not be explicitly described above. Features suitable for suchcombinations and sub-combinations would be readily apparent to personsskilled in the art upon review of the present application as a whole.The subject matter described herein and in the recited claims intends tocover and embrace all suitable changes in technology.

1. A biomimetic microfluidic system comprising: a substrate having afirst portion and a second portion; a first channel formed in the firstportion of the substrate, the first channel extending from a first inputto a first output along a longitudinal dimension, the first channelconfigured to selectively receive at least one first biologicalcomposition at a first channel flow rate, the at least one firstbiological composition including a biological source material; a secondchannel formed in the second portion of the substrate, the secondchannel extending from a second input to a second output along thelongitudinal dimension, the second channel configured to selectivelyreceive at least one second biological composition at a second channelflow rate, wherein at least a portion of the first and second channelsare substantially parallel; microchannels extending between the firstchannel and the second channel to create a fluid communication pathbetween the first and second channels, wherein the microchannels aresized to selectively capture the biological source material capable ofproducing target biological substances, wherein the first channel flowrate and the second channel flow rate are configured to selectivelycapture the biological source material at the microchannels and tocreate a differential between the first and second channels configuredto generate physiological shear rates along the second channel thatinduce the captured biological source material to produce the targetbiological substances.
 2. The system of claim 1, wherein themicrochannels are formed in at least one of a film, a membrane, or amesh arranged between the first and second channels.
 3. The system ofclaim 2, wherein the microchannels are formed as at least one of poresor slits extending between the first channel and the second channel. 4.The system of claim 1, wherein the biological source material includesmegakaryocytes and the target biological substances include platelets.5. The system of claim 1, wherein the first and second biologicalcompositions include at least one of a semi-solid, a solid, a liquid, ora collection of cells.
 6. The system of claim 1, wherein thephysiological shear rates are generated to be within a range between 100s⁻¹ and 10,500 s⁻¹.
 7. The system of claim 1, wherein the physiologicalshear rates are generated to be within a range between 500 s⁻¹ and 2,500s⁻¹ to induce a production of a plurality of platelets.
 8. The system ofclaim 1, wherein the physiological shear rates are controlled by atleast one of the first channel flow rate, the second channel flow rate,the longitudinal dimension, a transverse dimension of the first channel,a transverse dimension of the second channel, the first biologicalcomposition, or the second biological composition.
 9. The system ofclaim 1, wherein the system further comprises a first flow filterpositioned upstream from the first input, and a second flow filterpositioned upstream from the second input, wherein upstream is definedrelative to a direction of flow for the at least one first biologicalcomposition and the at least one second biological composition.
 10. Thesystem of claim 9, wherein the system further comprises a first flowresistor positioned between the first input and the first flow filter,and a second flow resistor positioned between the second input and thesecond flow filter.
 11. The system of claim 1, wherein the longitudinaldimension is between 1000 micrometers and 30,000 micrometers.
 12. Thesystem of claim 8, wherein the transverse dimensions of the first andsecond channels are between 100 micrometers and 3,000 micrometers. 13.The system of claim 1, wherein the microchannels are sized between 0.1micrometers and 20 micrometers.
 14. A method for generating a biologicalsubstance comprising: introducing a first biological composition into afirst channel of a biomimetic microfluidic system at a first channelflow rate, the at least one first biological composition including abiological source material capable of producing a target biologicalsubstance; introducing a second biological composition into a secondchannel of the biomimetic microfluidic system at a second channel flowrate; selectively capturing, by a series of microchannels forming afluid communication path between the first channel and the secondchannel, the biological source material; creating a differential betweenthe channels to generate physiological shear rates along the secondchannel that induce the captured biological source material to producethe target biological substance; and retrieving, using the secondbiological composition, the produced target biological substance forharvesting.
 15. The method of claim 14, wherein the microchannels aresized to permit capture of a plurality of platelet progenitors.
 16. Themethod of claim 14, wherein the first and second biological compositionsinclude at least one of a semi-solid, a solid, a liquid, or a collectionof cells.
 17. The method of claim 14, wherein the method furthercomprises creating the differential to generate physiological shearrates in a range between 500 s⁻¹ and 2,500 s⁻¹ to produce platelets fromcaptured megakaryocytes (MKs).
 18. The method of claim 14, wherein thephysiological shear rates are controlled by at least one of the firstchannel flow rate, the second channel flow rate, the longitudinaldimension, a transverse dimension of the first channel, a transversedimension of the second channel, the first biological composition, orthe second biological composition.
 19. The method of claim 14, whereinthe target biological substance comprises platelets.
 20. A method forgenerating platelets comprising: introducing a first biologicalcomposition into a first channel of a biomimetic microfluidic system ata first channel flow rate, the first biological composition includingmegakaryocytes (MKs) capable of generating platelets (PLTs); introducinga second biological composition into of the biomimetic microfluidicsystem at a second channel flow rate; selectively capturing, in thefirst channel, by a series of microchannels forming a fluidcommunication path between the first channel and the second channel, oneor more of the MKs; permitting pro-PLTs formed from the one or more MKsto extend through the microchannels into the second channel; creating adifferential between the channels to generate physiological shear ratesalong the second channel that induce production of the PLTs from thepro-PLTs; and retrieving, using the second biological composition, theproduced PLTs.